Protein-protein interactions are central to biological processes, determining the specificity of all cellular signaling events, and therefore represent an important class of drug targets. However, protein-protein interaction sites constitute large and flat interfaces (750-1500 Å) in each respective protein, rather than small hydrophobic, more ‘drugable’, pockets. Therefore, finding small-molecule inhibitors of protein-protein interaction has been proven to be a challenge (Arkin et al., 2004, Nat. Rev. Drug Discov., 3:301-317).
Members of the family of PKC isozymes are dependent on lipid-derived second messengers (as well as on calcium for some isozymes) that induce conformational changes, transforming the enzyme from an inactive to an active state. PKC activation is also associated with the translocation of the active enzymes to their partner proteins, RACKs (for receptor for activated C-Kinase), that facilitate the enzyme translocation (Ron et al., 1999, J. Biol. Chem., 274:27039-27046) to different sub-cellular sites (Mochly-Rosen, 1995, Science, 268:247-251). It was determined that the C2 domain in the regulatory region of PKC mediates at least some of the binding to their RACKs (Smith et al., 1992, Biochem. Biophys. Res. Commun., 188:1235-1240; Johnson et al., 1996, J. Biol. Chem., 271:24962-24966); unique sequences within the highly conserved C2 domain (e.g., βC2-4, δV1-1 and εV1-2 (Ron et al., 1995, J. Biol. Chem., 270:24180-24187; Chen et al., Proc. Natl. Acad. Sci. USA, 98:11114-11119; Gray et al., 1997, J. Biol. Chem., 272:30945-30951) in each PKC isozyme are part of these interaction sites. Peptides representing these unique sequences (e.g., δV1-1) serve as competitive inhibitors, inhibiting the association of the corresponding isozyme with its RACK and therefore inhibiting all the functions of a given isozyme. On the other hand, inhibitory intra-molecular protein-protein interactions keep the enzyme in the inactive state. It has been shown that at least one such intra-molecular interaction occurs between the RACK-binding site in PKC and a sequence in the enzyme that is homologous to its RACK, termed pseudo RACK (ΨRACK) (Dorn et al., 1999, Proc. Natl., Acad. Sci., 96:12798-12803). A peptide corresponding to this WRACK site competes with the intra-molecular inhibitory interaction, thus serving as a selective activator of the corresponding isozyme.
δPKC regulating peptides are known to play a significant role in cardiac ischemia and reperfusion injury (i.e., heart attack-induced injury). It has been previously shown that treatment after the ischemic event with ΨδRACK, the δPKC-specific activator, increased δPKC-mediated cardiac injury whereas treatment with δV1-1, the δPKC specific inhibitor, blocked this injury (Chen et al., Proc. Natl. Acad. Sci. USA, 98:11114-11119) in a variety of animal models of myocardial infarction, including mice and rats (Chen et al., Proc. Natl. Acad. Sci. USA, 98:11114-11119), pigs (Inagaki et al, 2003, Circulation, 108:2304-2307), and possibly humans (Bates et al., 2008, Circulation, 117:886-896).
Numerous substrates for δPKC have been identified in a variety of cell types and they are found in different sub-cellular locations. Further, it has been demonstrated that ischemia and reperfusion induce the translocation of some of the activated δPKC into the mitochondria (Churchill et al., 2005, Cir. Res., 97:78-85), leading to increased phosphorylation of the intra-mitochondrial enzyme, pyruvate dehydrogenase kinase (PDK). Phosphorylation of pyruvated dehydrogenase (PDH) by PDK results in decreased activity of PDH, thereby leading to the inhibition of the TCA cycle and ATP regeneration. Studies have suggested that cardiac efficiency and recovery of contractile function in postischemic hearts can be improved by pharmacological stimulation of PDH (Lewandowski et al., 1995, Circulation, 91:2017-2079; Schoder et al., 1998, Biochim Biophys. Acta., 1406:62-72). However, it was not clear whether phosphorylation of PDK alone by δPKC is responsible for resulting cardiac injury following ischemia and reperfusion (I/R). It is possible that any of the other δPKC substrates alone or together with PDK may contribute to or be critical for this injury.
To determine the importance of δPKC-mediated PDK phosphorylation for cardiac injury by I/R, a peptide inhibitor was designed that selectively inhibits PDK phosphorylation by δPKC without affecting the phosphorylation of other substrates of this isozyme. Since selective inhibitors and activators for PDK itself are not available, such a separation-of-function inhibitor of δPKC provides both an important tool to address the above question as well as the basis for a therapeutic composition for the treatment at least of tissue injury by ischemia and reperfusion.